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The field of cytometry involves the measurement of properties of individual biological cells suspended in a liquid medium. Cytometry uses optical properties of the cells themselves, such as light scattering properties, to identify and classify individual cells in a sample or fluorescent labels selectively attached to certain cells to further identify cells. Where fluorescent labels are used, multiple fluorescent labels may be used simultaneously, where each label can be distinguished by the spectral characteristics (color) of the light emitted or fluoresced by that label as well as the absorption and excitation of the label by illumination at different wavelengths.
Cytometry sometimes includes the measurement of a number of parameters of each cell in the sample. These parameters may include the size, morphology, cell type, health status (live or dead), deoxyribonucleic acid (“DNA”) content, and presence or absence of certain proteins or other molecules on the surface of the cells. A test may count the number of each type of cell in the sample, assay continuously variable properties of the cells in the sample, or do some combination of these types of tests. These are common examples and still other types of tests or analyses may be performed.
Another method of assaying biological content of a sample, termed here “binding assays”, uses microspheres made of polystyrene or other materials to capture and detect proteins or other biologically active molecules in a sample. Instead of measuring properties of individual cells, the microspheres are used to detect the presence or absence of biologically active compounds in the liquid sample. Capture molecules such as antibodies or nucleic acid sequences are attached to the outer surface of the microspheres, which then “capture” the target biologically active compound. The presence of the target compound in the sample may then be indicated using fluorescent labels which also bind to the target analyte, so that the microspheres indicate the presence and quantity of the analyte in the sample by the degree to which material bound to the surface of the microsphere fluoresces. Microsphere assays are able to identify proteins, compounds such as drugs of abuse, and specific nucleic acid sequences that may be present in the sample.
Flow cytometry is a technique within the field of cytometry that uses specially designed optically clear channels to present the particles (e.g., cells) in the sample one at a time to an optical system for measurement. The cells are typically illuminated by one or more focused lasers that illuminate only one cell at a time. The illumination may also be performed with other devices such as light emitting diodes (LEDs), are lamps, or other light sources.
Flow cytometry is an efficient means of evaluating a large number of cells in a sample since the time to measure each individual particle is on the order of a few microseconds. The properties that are typically recorded for each cell include forward scattered light, side scattered light, back-scattered light, and one or more colors of fluorescence used to identify the previously referenced fluorescent labels. A flow cytometer might use one, two, or more lasers to collect the desired number of measurements for each particle or cell in the sample.
Flow cytometry suffers a number of drawbacks. One drawback of flow cytometry results from measuring particles sequentially. In order to measure a large number of particles sequentially in a short period of time, the time allowed to measure each individual particle is also short. A second drawback results from the method of illumination typically employed in flow cytometers. In order to provide highly uniform illumination to each particle, whose position within the sample may vary from particle to particle, a field of illumination substantially larger than the particle is used. Typically, an illumination field ten times the diameter of each particle or greater is used illuminate each particle that only varies by a few percent. Consequently, flow cytometers are only able to use a small percentage of the illumination to analyze each particle. Because the illumination source is many times brighter than what is needed to illuminate a particle, the amount of stray light in the optical system is also much higher than desirable. Excess stray light interferes with the flow cytometer's ability to detect very weakly fluorescent particles.
Scanning cytometry, or laser-scanning cytometry, uses a microscope equipped with an optical scanning system to analyze and measure a number of cells or microspheres presented, for example, on a microscope slide for analysis. (Other presentation methods may also be used.) The samples are typically static; that is to say that particles being analyzed are spread out over a flat surface while being analyzed, and the optical system scans across the surface to evaluate the individual particles. Alternately, the slide holding the particles may be translated using a motorized stage beneath a fixed optical analysis system. Like a flow cytometer, a scanning cytometer is able to measure multiple fluorescence and light-scattering properties simultaneously.
Scanning cytometers address the illumination issues of flow cytometers by only illuminating the particle being analyzed with a focused light source (typically a laser). These instruments also can use lower power illumination sources and have substantially less stray light than flow cytometers.
Whereas a flow cytometer is able to measure an arbitrarily large number of particles for any sample, a scanning cytometer is typically limited by the area the machine can analyze (i.e., the field of view of the microscope). In order to enlarge the surface on which the particles are held, thereby increasing the number of particles that may be measured, scanning cytometers use precise translation stages that can move the surface through the field of view. This method of scanning increases the cost of the equipment and involves a long analysis time during which the scanning occurs.